There are several potential techniques for incorporating an isotope into a glycan. One method is to grow the cell or organism on a labeled material so that the isotope is included into all potential labeling sites in a cell, i.e., whole cell labeling. For instance, it is known in the art that mice grown on food that only contains 15N will result in all of the nitrogen in these mice being the isotopic variant, including the nitrogen in the sugars. Another similar approach is to grow cells/organisms with 13C-labeled glucose as its sole energy source. This has been done previously in the art with yeast, resulting in very high levels of isotope incorporation (Rampler et al., “Production of a 13C-labeled Internal Standard for Quantitative Glycomics,” proceedings of the 61st ASMS Conference on Mass Spectrometry, Minneapolis, Minn., June 2013).
However, whole-cell labeling is undesirable because it is expensive and the labeled glycans may not be of the correct type. In any type of isotopic labeling, the isotope is typically the most expensive reagent used in the technique, and in whole-cell labeling, the isotope goes into all molecules, not only glycans. Further, the most useful type of analytical standard contains molecules that are similar to, if not nearly identical to, the compound to be analyzed. In whole-cell labeling, the 15N mice and 13C yeast have different glycans than the glycans present in a recombinant IgG, for example. Thus, the advantage of using an isotopically labeled internal standard is lost.
Another potential method for isotopic glycan labeling is the use of isotopically-labeled nucleotide-sugars. An isotopic variant of a nucleotide sugar can be added to the cell culture media, e.g., UDP-GalNAc with the isotope label in the GalNAc. However, this approach is undesirable for several reasons: 1) Unlabeled nucleotide sugar would be present because they are continually biosynthesized by the cell, and consequently the extent of labeling is generally poor; 2) Nucleotide sugars are highly energetic and thus have short lifetimes in solutions, therefore, they would most likely need to be continually added to the media; 3) A mechanism for cells to incorporate nucleotide sugars into the cell where they are needed for glycoprotein biosynthesis is not readily available; and 4) Nucleotide sugars are difficult to obtain or synthesize. Accordingly, the use of isotopically-labeled nucleotide-sugars is not commercially viable.
Another potential method for the isotopic labeling of a glycan is by in vivo introduction of an isotope into a nucleotide sugar. To understand this method for incorporating isotopes into glycoprotein chains, the in vivo biosynthetic pathway of glycoprotein glycans must first be considered. The glycans attached to proteins are created by the sequential addition of multiple monosaccharides to either existing glycans (either N- or O-linked) or to the dolichol phosphate precursor for N-linked glycan chains. These monosaccharide additions are performed enzymatically, which controls the site of addition, and utilizes an activated nucleotide sugar as the source of the monosaccharide. For example, a glycosyltransferase would transfer a glucose residue from uracil-diphosphate glucose (UDP-glucose) to the glycan chain. Consequently, the addition of each different monosaccharide requires different transferases and different nucleotide sugars (UDP-GalNAc, GDP-Man, CMP-NeuAc, etc.)
Methods of isotopically labeling glycans via in vivo introduction of an isotope into a nucleotide sugar have been identified in the art for the purposes of identifying and/or quantifying glycans in a glycoprotein sample. For example, Wells et al. (U.S. Patent App. Pub. No. 2010/0297609, which is incorporated by reference herein in its entirety) describes a method of isotopically labeling glycans with 15N in cell culture for performing relative quantitative glycomics. The method described in Wells, termed isotopic detection of aminosugars with glutamine (IDAWG), relies on the hexosamine biosynthetic pathway that uses the side-chain of glutamine as the donor source of nitrogen for aminosugars in the production of sugar nucleotides. The IDAWG method is based on the introduction of 15N-glutamine into otherwise glutamine-free media to cause all aminosugars in cells or tissues grown in the media to become labeled, i.e., increased in mass by 1 dalton. The labeled cells or tissues can then be compared to control cells or tissues grown in 14N-glutamine media to perform glycan detection and/or quantification.
However, current methods of identifying and/or quantifying glycoprotein glycans, such as IDAWG, are associated with a level of error that is unacceptable for most applications. The average error associated with such methods has been found to be over 100% (Wada et al., 2007, Glycobiology, 17(4):411-422; Orlando et al., 2010, J Biomol Tech, 21(3 Suppl): S17-S18). This level of uncertainty is unacceptable for most, if not all, applications where glycans are desired to be identified and/or quantified. For example, a method with this level of uncertainty could not be used in applications needing FDA approval, such as the characterization of therapeutic agents.
In addition to the currently known glycan identification methods being associated with a high level of error, these methods are limited in potential end-use applications. For example, the IDAWG method is only useful for whole glycomic experiments, i.e., comparing one cell line to another cell line. Specifically, the IDAWG method involves the steps of growing a first cell line in a 15N-glutamine medium that is otherwise glutamine-free, while growing a second cell line, or control line, in a 14N-glutamine medium. The first cell line can be manipulated in some manner during growth, for example by treating the cell line with one or more chemicals or by varying environmental conditions, and the effects of the manipulation can be determined by comparing the isolated glycoconjugates or glycans from the first cell line to the isolated glycoconjugates or glycans from the control cell line via mass spectrometry.
However, the labeling techniques in the IDAWG method cannot generally be used to create a standard comprising glycans or glycoconjugates that can be used to analyze a sample from a different cell line because the extent of isotopic labeling by the IDAWG method is typically not high enough to provide accurate quantification of glycans outside of a direct comparison of two cell lines. Accordingly, the IDAWG method is only useful for a relative, but not absolute, analysis of glycans.
Thus, there is a continuing need for new and/or improved methods of isotopically labeling glycans, and for new methods of identifying and quantifying glycans. In addition, there is a need in the art for producing isotopically labeled glycans or glycoproteins for use as an analytical reference standard. The present invention addresses these continuing needs in the art.